Arabidopsis tic62 trol Mutant Lacking Thylakoid- Bound Ferredoxin-NADP＋ Oxidoreductase Shows Distinct Metabolic Phenotype

Ferredoxin-NADP＋ oxidoreductase （FNR）, functioning in the last step of the photosynthetic electron transfer chain, exists both as a soluble protein in the chloroplast stroma and tightly attached to chloroplast membranes. Surface plasmon resonance assays showed that the two FNR isoforms, LFNR1 and LFNR2, are bound to the thylakoid membrane via the C-terminal domains of Tic62 and TROL proteins in a pH-dependent manner. The tic62 trol double mutants contained a reduced level of FNR, exclusively found in the soluble stroma. Although the mutant plants showed no visual phenotype or defects in the function of photosystems under any conditions studied, a low ratio of NADPH/NADP~ was detected. Since the CO2 fixation capacity did not differ between the tic62 trol plants and wild-type, it seems that the plants are able to funnel reducing power to most crucial reactions to ensure survival and fitness of the plants. However, the activity of malate dehydrogenase was down-regulated in the mutant plants. Apparently, the plastid metabolism is able to cope with substantial changes in directing the electrons from the light reactions to stromal metabolism and thus only few differences are visible in steady-state metabolite pool sizes of the tic62 trol plants.     

Cell Wall,Cytoskeleton, and Cell Expansion in Higher Plants

To accommodate two seemingly contradictory biological roles in plant physiology, providing both the rigid structural support of plant cells and the adjustable elasticity needed for cell expansion, the composition of the plant cell wall has evolved to become an intricate network of cellulosic, hemicellulosic, and pectic polysaccharides and protein. Due to its complexity, many aspects of the cell wall influence plant cell expansion, and many new and insightful observations and technologies are forthcoming. The biosynthesis of cell wall polymers and the roles of the variety of proteins involved in polysaccharide synthesis continue to be characterized. The interactions within the cell wall polymer network and the modification of these interactions provide insight into how the plant cell wall provides its dual function. The complex cell wall architecture is controlled and organized in part by the dynamic intracellular cytoskeleton and by diverse trafficking pathways of the cell wall polymers and cell wall-related machinery. Meanwhile, the cell wall is continually influenced by hormonal and integrity sensing stimuli that are perceived by the cell. These many processes cooperate to construct, maintain, and manipulate the intricate plant cell wall--an essential structure for the sustaining of the plant stature, growth, and life.     

Import Determinants of Organelle-Specific and Dual Targeting Peptides of Mitochondria and Chloroplasts in Arabidopsis thaliana

Most of the mitochondrial and chloroplastic proteins are synthesized in the cytosol as precursor proteins carrying an N-terminal targeting peptide （TP） directing them specifically to a correct organelle. However, there is a group of proteins that are dually targeted to mitochondria and chloroplasts using an ambiguous N-terminal dual targeting peptide （dTP）. Here, we have investigated pattern properties of import determinants of organelle-specific TPs and dTPs combining mathematical multivariate data analysis （MVDA） with in vitro organellar import studies. We have used large datasets of mitochondrial and chloroplastic proteins found in organellar proteomes as well as manually selected data sets of experimentally confirmed organelle-specific TPs and dTPs from Arabidopsis thaliana. Two classes of organelle-specific TPs could be distinguished by MVDA and potential patterns or periodicity in the amino acid sequence contributing to the separation were revealed, dTPs were found to have intermediate sequence features between the organelle-specific TPs. Interestingly, introducing positively charged residues to the dTPs showed clustering towards the mitochondrial TPs in silico and resulted in inhibition of chloroplast, but not mitochondrial import in in vitro organellar import studies. These findings suggest that positive charges in the N-terminal region of TPs may function as an ＇avoidance signal＇ for the chloroplast import.     

Dissecting the Heat Stress Response in Chlamydomonas by Pharmaceutical and RNAi Approaches Reveals Conserved and Novel Aspects

To study how conserved fundamental concepts of the heat stress response （HSR） are in photosynthetic eukaryotes, we applied pharmaceutical and antisense/amiRNA approaches to the unicellular green alga Chlamydomonas reinhardtii. The Chlamydomonas HSR appears to be triggered by the accumulation of unfolded proteins, as it was induced at ambient temperatures by feeding cells with the arginine analog canavanine. The protein kinase inhibitor staurosporine strongly retarded the HSR, demonstrating the importance of phosphorylation during activation of the HSR also in Chlamydomonas. While the removal of extracellular calcium by the application of EGTA and BAPTA inhibited the HSR in moss and higher plants, only the addition of BAPTA, but not of EGTA, retarded the HSR and impaired thermotoler- ance in Chlamydomonas. The addition of cycloheximide, an inhibitor of cytosolic protein synthesis, abolished the attenu- ation of the HSR, indicating that protein synthesis is necessary to restore proteostasis. HSP90 inhibitors induced a stress response when added at ambient conditions and retarded attenuation of the HSR at elevated temperatures. In addition, we detected a direct physical interaction between cytosolic HSP90A/HSP70A and heat shock factor 1, but surprisingly this interaction persisted after the onset of stress. Finally, the expression of antisense constructs targeting chloroplast HSP70B resulted in a delay of the cell＇s entire HSR, thus suggesting the existence of a retrograde stress signaling cascade that is desensitized in HSP7OB-antisense strains.     

Control of Rice Embryo Development,Shoot Apical Meristem Maintenance,and Grain Yield by a Novel Cytochrome P450

Angiosperm seeds usually consist of two major parts： the embryo and the endosperm. However, the molec- ular mechanism（s） underlying embryo and endosperm development remains largely unknown, particularly in rice, the model cereal. Here, we report the identification and functional characterization of the rice GIANT EMBRYO （GE） gene. Mutation of GE resulted in a large embryo in the seed, which was caused by excessive expansion of scuteUum cells. Post-embryonic growth of ge seedling was severely inhibited due to defective shoot apical meristem （SAM） mainte- nance. Map-based cloning revealed that GE encodes a CYP78A subfamily P450 monooxygenase that is localized to the endoplasmic reticulum. GE is expressed predominantly in the scutellar epithelium, the interface region between embryo and endosperm. Overexpression of GE promoted cell proliferation and enhanced rice plant growth and grain yield, but reduced embryo size, suggesting that GE is critical for coordinating rice embryo and endosperm development. Moreover, transgenic Arabidopsis plants overexpressing AtCYP78AlO, a GE homolog, also produced bigger seeds, implying a con- served role for the CYP78A subfamily of P450s in regulating seed development. Taken together, our results indicate that GE plays critical roles in regulating embryo development and SAM maintenance.     

Redox Regulation of Arabidopsis Mitochondrial Citrate Synthase

Citrate synthase has a key role in the tricarboxylic （TCA） cycle of mitochondria of all organisms, as it cata- lyzes the first committed step which is the fusion of a carbon-carbon bond between oxaloacetate and acetyl CoA. The regulation of TCA cycle function is especially important in plants, since mitochondrial activities have to be coordinated with photosynthesis. The posttranslational regulation of TCA cycle activity in plants is thus far almost entirely unexplored. Although several TCA cycle enzymes have been identified as thioredoxin targets in vitro, the existence of any thioredoxin-dependent regulation as known for the Calvin cycle, yet remains to be demonstrated. Here we have investigated the redox regulation of the Arabidopsis citrate synthase enzyme by site-directed mutagenesis of its six cysteine residues. Our results indicate that oxidation inhibits the enzyme activity by the formation of mixed disulfides, as the partially oxidized citrate synthase enzyme forms large redox-dependent aggregates. Furthermore, we were able to demonstrate that thioredoxin can cleave diverse intraas well as intermolecular disulfide bridges, which strongly enhances the activity of the enzyme. Activity measurements with the cysteine variants of the enzyme revealed important cysteine residues affecting total enzyme activity as well as the redox sensitivity of the enzyme.     

Organelle pH in the Arabidopsis Endomembrane System

The pH of intracellular compartments is essential for the viability of cells. Despite its relevance, little is known about the pH of these compartments. To measure pH in vivo, we have first generated two pH sensors by combining the improved-solubility feature of solubility-modified green fluorescent protein （GFP） （smGFP） with the pH-sensing capabil- ity of the pHluorins and codon optimized for expression in Arabidopsis. PEpHluorin （plant-solubility-modified ecliptic pHluorin） gradually loses fluorescence as pH is lowered with fluorescence vanishing at pH 6.2 and PRpHluorin （plant- solubility-modified ratiomatric pHluorin）, a dual-excitation sensor, allowing for precise measurements. Compartment- specific sensors were generated by further fusing specific sorting signals to PEpHluorin and PRpHluorin. Our results show that the pH of cytosol and nucleus is similar （pH 7.3 and 7.2）, while peroxisomes, mitochondrial matrix, and plastidial stroma have alkaline pH. Compartments of the secretory pathway reveal a gradual acidification, spanning from pH 7.1 in the endoplasmic reticulum （ER） to pH 5.2 in the vacuole. Surprisingly, pH in the trans-Golgi network （TGN） and mul- tivesicular body （MVB） is, with pH 6.3 and 6.2, quite similar. The inhibition of vacuolar-type H＋-ATPase （V-ATPase） with concanamycin A （ConcA） caused drastic increase in pH in TGN and vacuole. Overall, the PEpHluorin and PRpHluorin are excellent pH sensors for visualization and quantification of pH in vivo, respectively.     

How does the host-specialized aphid deal with food deficiency？

Aphis gossypii Glover shows obvious host specialization, with cucurbit- and cotton-specialized biotypes or host races in many regions. Because its annual natal hostcrops senesce earlier the cucurbit-specialized biotype may suffer food deficiency. The method this biotype uses to overcome this challenge is still poorly understood. In orderto understand the potential of the cucurbit-specialized biotype aphids in host shift and usage, the performance of this biotype on cotton （Gossypium hirsutum）, a common butpoor quality host plant, was explored in this study. The cucurbit-specialized aphids could establish populations on cotton only when these plants had at least nine leaves, and subsequent populations developed rather slowly. The presence of whitefly populations on cotton improved the success rate of cucurbit-specialized aphids. The cucurbit-specialized aphidswere mainly distributed on the older leaves of cotton, with only a few settling on the upper leaves. The cucurbit-specialized aphids reared on cotton for 40, 54 and 61 days stillmaintained strong preference for their natal host plant, cucumber （Cucumis sativus）, rather than cotton, and their net reproductive rates and intrinsic rates of natural increase weredramatically lower when they were transferred onto new six-leaf cotton plants or detached leaves. Therefore, we concluded that the cucurbit-specialized aphids have the potentialto utilize mature or whitefly-stressed cotton plants, but that this feeding experience on cotton did not alter their specialization for cucurbits. Some cotton plants could act as atemporary host for the cucurbit-specialized aphids to overcome food deficiency arising from senescing cucurbits.     

Plastid Signals and the Bundle Sheath： Mesophyll Development in Reticulate Mutants

The development of a plant leaf is a meticulously orchestrated sequence of events producing a complex organ comprising diverse cell types. The reticulate class of leaf variegation mutants displays contrasting pigmentation between veins and interveinal regions due to specific aberrations in the development of mesophyll cells. Thus, the reticulate mutants offer a potent tool to investigate cell-type-specific developmental processes. The discovery that most mutants are affected in plastid-localized, metabolic pathways that are strongly expressed in vasculature-associated tis- sues implicates a crucial role for the bundle sheath and their chloroplasts in proper development of the mesophyll cells. Here, we review the reticulate mutants and their phenotypic characteristics, with a focus on those in Arabidopsis thali- ana. Two alternative models have been put forward to explain the relationship between plastid metabolism and meso- phyll cell development, which we call here the supply and the signaling hypotheses. We critically assess these proposed models and discuss their implications for leaf development and bundle sheath function in C3 species. The characteriza- tion of the reticulate mutants supports the significance of plastid retrograde signaling in cell development and highlights the significance of the bundle sheath in C3 photosynthesis.     

眶额叶区5-HT与Glu、NO在急性强迫游泳应激抑郁症模型中的关系

目的：探讨眶额叶区5-羟色胺（5-HT）与谷氨酸（Glu）、一氧化氮（N0）在急性强迫游泳应激抑郁症模型中的相互作用。方法：雄性SD大鼠随机分为对照组及各种药物注射组,强迫游泳制造大鼠应激性抑郁模型,眶额叶区微量注射各组药物,敞箱实验及游泳测试观察大鼠的抑郁样行为表现。结果：①与对照组比,注射Glu使大鼠强迫游泳不动时间显著增加;注射NMDA受体拮抗剂（MK-801）使大鼠强迫游泳不动时间减少;与Glu组比,MK-801预注射后Glu注射使大鼠强迫游泳不动时间减少;②与5-HT组比,MK-801预注射后5-HT注射使大鼠强迫游泳不动时间增加;③与对照组比,注射L-精氨酸（L-Ars）使大鼠强迫游泳不动时间显著增加;注射NOS抑制剂（L-NAME）（10μg/μl）使大鼠强迫游泳不动时间减少;L-NAME（20μg／μl）注射使大鼠强迫游泳不动时间增加;L-NAME（40μg/μl）注射使大鼠强迫游泳不动时间增加;④与L-NAME（10μg/μl）组比较,5-HT1A受体拮抗剂spipemne预注射后LNAME（10μg／μl）注射使大鼠强迫游泳不动时间增加。结论：眶额叶（OFC）区Glu含量的增加能够诱发抑郁,其作用可能主要是通过NMDA受体实现的,Glu经NMDA受体引发抑郁的同时还可能通过调节突触后膜上5-HT1A受体减弱5-HT的抗抑郁作用;OFC区NO可通过调节5-HT神经元进而参与抑郁的发生。     

Aphis （Hemiptera： Aphididae） species groups found in the Midwestern United States and their contribution to the phylogenetic knowledge of the genus

A phylogeny of the genus Aphis Linnaeus, 1 758 was built primarily from specimens collected in the Midwest of the United States. A data matrix was constructedwith 68 species and 41 morphological characters with respective character states of alate and apterous viviparous females. Dendrogram topologies of analyses performed usingUPGMA （Unweighted Pair Group Method with Arithmetic Mean）, Maximum Parsimony and Bayesian analysis of Cytochrome Oxidase I, Elongation Factor 1-α and primary endosymbiont Buchnera aphidicola 16S sequences were not congruent. Bayesian analysis strongly supported most terminal nodes of the phylogenetic trees. The phylogeny wasstrongly supported by EFI-α, and analysis of COl and EFI-α molecular data combined with morphological characters. It was not supported by single analysis of COI or Buch-hera aphidicola 16S. Results from the Bayesian phylogeny show 4 main species groups： asclepiadis,fabae, gossypii, and middletonii. Results place Aphis and species of the generaProtaphis Bōrner, 1952, Toxoptera Koch, 1856 and Xerobion Nevsky, 1928 in a monophyletic clade. Morphological characters support this monophyly as well. The phylogenyshows that the monophyletic clade of the North American middletonii species group belong to the genus Protaphis： P. debilicornis （Gillette ＆ Palmer, 1929）, comb. nov., P. echinaceae（Lagos and Voegtlin, 2009）, comb. nov., and P. middletonii （Thomas, 1879）. The genus Toxoptera should be considered a subgenus of Aphis （stat. nov.）. The analysis also indicatesthat the current genus Iowana Frison, 1954 should be considered a subgenus of Aphis （stat. nov.）.     

Heteromeric and Homomeric Geranyl Diphosphate Synthases from Catharanthus roseus and Their Role in Monoterpene Indole Alkaloid Biosynthesis

Catharanthus roseus is the sole source of two most important monoterpene indole alkaloid （MIA） anti- cancer agents： vinblastine and vincristine. MIAs possess a terpene and an indole moiety derived from terpenoid and shikimate pathways, respectively. Geranyl diphosphate （GPP）, the entry point to the formation of terpene moiety, is a product of the condensation of isopentenyl diphosphate （IPP） and dimethylallyl diphosphate （DMAPP） by GPP synthase （GPPS）. Here, we report three genes encoding proteins with sequence similarity to large subunit （CrGPPS.LSU） and small subunit （CrGPPS.SSU） of heteromeric GPPSs, and a homomeric GPPSs. CrGPPS.LSU is a bifunctional enzyme producing both GPP and geranyl geranyl diphosphate （GGPP）, CrGPPS.SSU is inactive, whereas CrGPPS is a homomeric enzyme forming GPP. Co-expression of both subunits in Escherichia coil resulted in heteromeric enzyme with enhanced activity producing only GPR While CrGPPS.LSU and CrGPPS showed higher expression in older and younger leaves, respectively, CrGPPS.SSU showed an increasing trend and decreased gradually. Methyl jasmonate （MelA） treatment of leaves sig- nificantly induced the expression of only CrGPPS.SSU. GFP localization indicated that CrGPPS.SSU is plastidial whereas CrGPPS is mitochondrial. Transient overexpression of AmGPPS.SSU in C. roseus leaves resulted in increased vindoline, immediate monomeric precursor of vinblastine and vincristine. Although C. roseus has both heteromeric and homomeric GPPS enzymes, our results implicate the involvement of only heteromeric GPPS with CrGPPS.SSU regulating the GPP flux for MIA biosynthesis.     

Regulation of Cytokinesis by Exocyst Subunit SEC6 and KEULE in Arabidopsis thaliana

Proper vesicle tethering and membrane fusion at the cell plate are essential for cytokinesis. Both the vesicle tethering complex exocyst and membrane fusion regulator KEULE were shown to function in cell plate formation, but the exact mechanisms still remain to be explored. In this study, using yeast two-hybrid （Y-2-H） assay, we found that SEC6 interacted with KEULE, and that a small portion of C-terminal region of KEULE was required for the interaction. The direct SEC6-KEULE interaction was supported by further studies using in vitro pull-down assay, immunoprecipitation, and in vivo bimolecular florescence complementation （BIFC） microscopy, sec6 mutants were male gametophytic lethal as reported; however, pollen-rescued sec6 mutants （PRsec6） displayed cytokinesis defects in the embryonic cells and later in the leaf pavement cells and the guard cells. SEC6 and KEULE proteins were co-localized to the cell plate during cytokine- sis in transgenic Arabidopsis. Furthermore, only SEC6 but not other exocyst subunits located in the cell plate interacted with KEULE in vitro. These results demonstrated that, like KEULE, SEC6 plays a physiological role in cytokinesis, and the SEC6-KEULE interaction may serve as a novel molecular linkage between arriving vesicles and membrane fusion machin- ery or directly regulate membrane fusion during cell plate formation in plants.     

Perturbation of Auxin Homeostasis Caused by Mitochondrial FtSH4 Gene-Mediated Peroxidase Accumulation Regulates Arabiclopsis Architecture

Reactive oxygen species and auxin play important roles in the networks that regulate plant development and morphogenetic changes, However, the molecular mechanisms underlying the interactions between them are poorly understood. This study isolated a mas （More Axillary Shoots） mutant, which was identified as an allele of the mitochondrial AAA-protease AtFtSH4, and characterized the function of the FtSH4 gene in regulating plant development by medi- ating the peroxidase-dependent interplay between hydrogen peroxide （H2Oz） and auxin homeostasis. The phenotypes of dwarfism and increased axillary branches observed in the mas （renamed as ftsh4-4） mutant result from a decrease in the IAA concentration. The expression levels of several auxin signaling genes, including IAA1, IAA2, and IAA3, as well as several auxin binding and transport genes, decreased significantly in ftsh4-4 plants. However, the H202 and peroxidases levels, which also have IAA oxidase activity, were significantly elevated in ftsh4-4 plants. The ftsh4-4 phenotypes could be reversed by expressing the iaaM gene or by knocking down the peroxidase genes PRX34 and PRX33. Both approaches can increase auxin levels in the ftsh4-4 mutant. Taken together, these results provided direct molecular and genetic evidence for the interaction between mitochondrial ATP-dependent protease, H2O2, and auxin homeostasis to regulate plant growth and development.     

Genotoxicity biomarkers in aquatic bioindicators

Pollution of the aquatic environment is an ever-growing problem, as waters are the ultimate sink for the large number of xenobiotics from multiple sources. DNA damaging agents have a significant ecological relevance since they are implicated in many pathological processes and exert effects beyond that of individual being active through following generations. A large number of methods have been applied to evaluate genotoxic damage in different aquatic species. Comet assay, as method for de- tecting DNA alterations, and micronucleus test, as an index of chromosomal damage are the most widely applied and validated methods in field studies. These methods were applied in different vertebrate and invertebrate aquatic species, but only mollusk and fish species have been employed in routine biomonitoring programs. Mussels, due to their widely geographical distribution and the suitability for caging represent the bioindicator of choice in field studies. Mytilus species is the most used marine mussel. The use of fish is limited to specific geographic areas. The present review mainly focuses on the application of comet assay and micronucleus test in mussels. A number of biomonitoring studies in mussels, using comet assay or micronucleus test, revealed exposure to different classes of genotoxic compounds with a good discrimination power. The different evidence from the two as- says, reflects different biological mechanisms for the two genetic endpoints, DNA damage and chromosomal damage, suggesting their combined application in the field. Different endogenous and exogenous factors have been shown to modulate the genotoxic responses in mussels, acting as confounding factors in environmental monitoring. The use of standardized protocol for caging, sampling and genotoxity evaluation is critical in biomonitoring studies. The use of a multimarker approach coupling genotoxicity biomarkers with physiological and biochemical factors allows to have a complete picture of the environmental pollution [Current Zoology 60 （2）： 273-284, 2014].     

Knights in Action： Lectin Receptor-Like Kinases in Plant Development and Stress Responses

The Receptor-Like Kinase （RLK） is a vast protein family with over 600 genes in Arabidopsis and 1100 in rice. The Lectin RLK （LecRLK） family is believed to play crucial roles in saccharide signaling as well as stress perception. All the LecRLKs possess three domains： an N-terminal lectin domain, an intermediate transmembrane domain, and a C-terminal kinase domain. On the basis of lectin domain variability, LecRLKs have been subgrouped into three subclasses： L-, G-, and C-type LecRLKs. While the previous studies on LecRLKs were dedicated to classification, comparative structural analysis and expression analysis by promoter-based studies, most of the recent studies on LecRLKs have laid special emphasis on the potential of this gene family in regulating biotic/abiotic stress and developmental pathways in plants, thus mak- ing the prospects of studying the LecRLK-mediated regulatory mechanism exceptionally promising. In this review, we have described in detail the LecRLK gene family with respect to a historical, evolutionary, and structural point of view. Furthermore, we have laid emphasis on the LecRLKs roles in development, stress conditions, and hormonal response. We have also discussed the exciting research prospects offered by the current knowledge on the LecRLK gene family. The multitude of the LecRLK gene family members and their functional diversity mark these genes as both interesting and worthy candidates for further analysis, especially in the field of crop improvement.     

A New Set of Reversibly Photoswitchable Fluorescent Proteins for Use in Transgenic Plants

Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state in response to specific wavelengths of light are novel tools for monitoring of protein trafficking and super-resolu- tion fluorescence microscopy in living organisms. Here, we describe variants of the reversibly photoswitchable fluores- cent proteins rsFastLime, bsDronpa, and Padron that have been codon-optimized for the use in transgenic Arabidopsis plants. The synthetic proteins, designated rsFastLIME-s, bsDRONPA-s, and PADRON C-s, showed photophysical properties and switching behavior comparable to those reported for the original proteins. By combining the ＇positively switchable＇ PADRON C-s with the ＇negatively switchable＇ rsFastLIME-s or bsDRONPA-s, two different fluorescent reporter proteins could be imaged at the same wavelength upon transient expression in Nicotiana benthamiana cells. Thus, co-localiza- tion analysis can be performed using only a single detection channel. Furthermore, the proteins were used to tag the RNA-binding protein AtGRP7 （Arabidopsis thaliana glycine-rich RNA-binding protein 7） in transgenic Arabidopsis plants. Because the new reversibly photoswitchable fluorescent proteins show an increase in signal strength during each pho- toactivation cycle, we were able to generate a large number of scans of the same region and reconstruct 3-D images of AtGRP7 expression in the root tip. Upon photoactivation of the AtGRP7：rsFastLIME-s fusion protein in a defined region of a transgenic Arabidopsis root, spreading of the fluorescence signal into adjacent regions was observed, indicating that movement from cell to cell can be monitored. Our results demonstrate that rsFastLIME-s, bsDRONPA-s, and PADRON C-s are versatile fluorescent markers in plants, Furthermore, the proteins also show strong fluorescence in mammalian cells including COS-7 and HeLa cells.